The gram stain is the most widely used taxonomic test of bacteria See P. Gerhardt, ed., Manual of Methods for General Bacteriology (4th ed. 1981). The technique is relatively simple, and in experienced hands gives reproducible results. The existing techniques do, however, have limitations. Most modifications of the gram stain, such as Hucker's, see G. Hucker, J. Bact. 6, 395 (1921), require at least four solutions and our staining steps. Furthermore, the stains used in the technique, particularly the primary stain, crystal violet, are concentrated and can be messy. The procedure is time consuming, with a single stain estimated to take approximately three minutes. K. Appel and M. Buschle, Lab. Med. 9, 149 (1985).
A variety of factors can produce erroneous results when using the conventional gram stain. See generally J. Bartholomew and T. Mittwer, Bact. Rev. 16, 1 (1953), K. Appel and M. Buschle, supra. P. Gerhardt, supra. For example, gram negative bacteria will appear as gram positive if decolorizing is incomplete or slide preparation results in a thick smear. Gram positive bacteria can also give false negative staining reactions, particularly when an old culture is used or decolorization is too extensive. Fortunately, these shortcomings are usually compensated for by the experience of bacteriologists. For as every instructor of bacteriology knows, training is essential for proper execution and interpretation of the gram stain.
Accordingly, objects of the present invention are to provide a new gram staining procedure which is simple, rapid, easy to interpret, resistant to errors, and requires few reagents.